Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coli

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ (L36), a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. Knockout of ZntA, a zinc efflux pump, abolished the zinc-resistant phenotype of the rpmJ mutant. RNA sequence analysis revealed that the rpmJ mutant exhibited altered gene expression of diverse functional categories, including translation, energy metabolism, and stress response. These findings suggest that knocking out RpmJ alters gene expression patterns and causes zinc resistance by lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance.


Introduction
Zinc is an essential metal for organisms. Approximately 5% to 6% of total proteins in bacteria are zinc-binding proteins [1]. Zinc acts as a cofactor for enzyme activity and protein structure folding. On the other hand, excess zinc is toxic to cells by destroying [4Fe-4S] clusters of dehydratases and releasing free irons [2]. Iron, a metal with high redox potential, produces reactive oxygen species by the Fenton-reaction and impairs cell growth [2][3][4].
Bacteria must maintain a strict intracellular zinc concentration to reserve a necessary amount of zinc while avoiding toxicity from excess zinc. Four main zinc transporters have been identified in Escherichia coli. ZnuABC [5], a high-affinity ABC transporter, and ZupT [6], a ZIP family transporter, are responsible for zinc uptake. Under zinc-deficient conditions, the expression of ZnuABC is upregulated by relieving the transcriptional repressor Zur, a homolog of Fur [5]. ZntA, a P-type ATPase transporter [7,8], and ZitB, a cation diffusion facilitator family transporter, mediate zinc efflux [9]. Under excess zinc conditions, the transcription factor ZntR upregulates the expression of ZntA [10,11]. Other than the zinc efflux and uptake systems, little is currently known about the factors involved in zinc resistance. In the present study, we aimed to identify the genetic factors responsible for zinc resistance utilizing a gene knockout mutant E. coli library. We found that knockout of the 50S ribosomal protein RpmJ (L36) conferred zinc resistance. The E. coli ribosome contains 54 proteins, of which RpmJ is 1 of 8 nonessential ribosomal proteins. RpmJ is the smallest 50S ribosomal protein with only 38 amino acids [12], and is involved in 23S rRNA folding [13]. We investigated the mechanism of zinc resistance in the rpmJ knockout mutant by analyzing gene expression and intracellular zinc concentration.

Knockout of rpmJ causes zinc resistance
In this study, we searched a gene knockout mutant library for gene knockout mutants that grew on Luria broth (LB) agar plates containing 1.4 mM zinc to identify genes whose deletions confer zinc resistance to E. coli. Four zinc-resistant mutant strains were identified (Table 1) with the rpmJ mutant exhibiting the strongest zinc-resistant phenotype ( Fig 1A). The MIC of wild-type against zinc was 1.4 mM and that of the rpmJ mutant was 2.0 mM. The other 3 mutant strains were pitA, rimP, and tufA mutants. PitA functions as a zinc uptake system [14], RimP is required for 30S ribosome maturation [15], and Elongation factor Tu1 (tufA) is required for ribosomal peptide elongation [16].
We performed a complementation test to confirm that zinc resistance is caused by a lack of rpmJ. The results demonstrated that introducing the rpmJ gene into the rpmJ mutant reduced the zinc resistance ( Fig 1B). In contrast, zinc resistance was not reduced by introducing mutated rpmJ genes in which C27 or H33, important amino acids for the zinc-finger structure of RpmJ [13,17], were replaced with serine ( Fig 1B). These results indicate that the loss of RpmJ function by destroying the zinc-finger structure leads to zinc resistance in E. coli.

Knockout of rpmJ alters ribosomal function
Given that RpmJ is a ribosomal protein, its knockout could alter the ribosomal structure. We examined the sensitivity of the rpmJ mutant to protein synthesis inhibitors that target ribosomes. Compared with the wild-type strain, the growth of the rpmJ mutant was decreased by all 4 tested inhibitors, chloramphenicol, erythromycin, clarithromycin, and tetracycline ( Fig  2). This finding implies that the rpmJ mutant has altered translation activity. Then, we focused on the translational function of the ribosome, and measured the translation fidelity using a dual luciferase assay in which stop codon readthroughs or frameshift readthroughs were detected ( Fig 3A) [18]. In the assay, stop codons or frameshift mutations are inserted between Rluc and Fluc genes, and a low Fluc/Rluc (F/R) value indicates that the translation is accurate [18]. In the UGA stop codon readthrough, the F/R values were higher in the rpmJ mutant than in the wild-type strain in both the no-zinc and 0.8-mM zinc conditions (Fig 3B). In the UAG stop codon readthrough, difference of the F/R values was not detected between the wild-type strain and the rpmJ mutant in the no-zinc and 0.8-mM zinc conditions ( Fig 3B). In the +1 frameshift readthrough, the F/R value was higher in the rpmJ mutant than in the wild-type strain in the no-zinc condition, but no difference was observed in the 0.8-mM zinc condition ( Fig 3B). In the -1 frameshift readthrough, difference of the F/R value was not detected between the wild-type strain and the rpmJ mutant in the no-zinc condition, but the F/R value was lower in the rpmJ mutant than in the wild-type strain in the 0.8-mM zinc condition ( Fig  3B). These results suggest that the ribosomal function required to maintain translation fidelity was altered in the rpmJ mutant.

The rpmJ mutant has a low intracellular zinc concentration under excess zinc conditions
The ability of the rpmJ mutant to grow in an excess zinc condition could be due to a low intracellular zinc concentration. We measured the intracellular zinc concentration by inductively coupled plasma-mass spectrometry (ICP-MS) [19]. In a no-zinc and a 0.6-mM zinc conditions, the intracellular zinc concentrations did not differ between the wild-type strain and rpmJ mutant (Fig 4). In a 1.2-mM excess zinc condition, the intracellular zinc concentration was lower in the rpmJ mutant than in the wild-type strain, but there was no significant difference between the wild-type strain and the rpmJ mutant transformed with the intact rpmJ gene ( Fig 4). These results suggest that the rpmJ mutant maintained a low intracellular zinc concentration under an excess zinc condition, which could confer zinc resistance to the rpmJ mutant.

Knockout of rpmJ alters global gene expression patterns
To understand the molecular mechanisms underlying the zinc resistance of the rpmJ mutant, we performed RNA sequence analysis to identify differentially expressed genes in the rpmJ mutant. In the rpmJ mutant, 195 genes were upregulated and 275 genes were downregulated compared with the wild-type strain (S1 Table). Contrary to our expectation, the expression of zinc uptake or zinc efflux genes was not altered in the rpmJ mutant. In contrast, expression of 6 genes encoding synthases for iron-sulfur clusters was decreased in the rpmJ mutant (S1 Table). Because iron-sulfur clusters are toxic targets of zinc, decreased amounts of iron-sulfur clusters could contribute to the zinc resistance of the rpmJ mutant. To elucidate characteristic features of the differentially expressed genes in the rpmJ mutant, we performed a gene ontology (GO) enrichment analysis. The upregulated genes included those categorized as related to translation or ribosomal subunits (Fig 5A), suggesting that ribosomal function is damaged in the rpmJ knockout and some compensatory regulatory mechanisms were triggered to increase translation function. The genes related to aerobic ATP synthesis were found in upregulated genes ( Fig 5A). The downregulated genes included those categorized as related to anaerobic respiration, stress response, amino acid metabolism, glycogen metabolism ( Fig 5B).

The zntA gene is required for the zinc resistance caused by the rpmJ knockout
Although the RNA sequence analysis suggest that the expression of zinc uptake or zinc efflux genes was not altered in the rpmJ mutant at the transcript level in the absence of zinc, there are still possibilities that the rpmJ knockout alters the expression of zinc uptake or zinc efflux genes in the presence of zinc, and decrease the zinc concentration. We examined whether the zntA and zitB genes that encode zinc efflux pumps are involved in the zinc resistance of the rpmJ mutant by analyzing zinc resistance phenotype of gene knockout mutants. The zntA knockout mutant was sensitive to zinc compared with the wild-type strain (Fig 6A).
The double knockout mutant of rpmJ and zntA was sensitive to zinc, whose growth was comparable with that of the zntA knockout mutant (Fig 6A). In contrast, in the absence of zinc, the zntA mutant and rpmJ/zntA double knockout mutant showed indistinguishable growth from the wild-type strain (Fig 6A). The growth of the zitB knockout mutant was indistinguishable from that of the wild-type strain in the presence of zinc (Fig 6B). The doble knockout mutant of rpmJ and zitB exhibited indistinguishable growth with the rpmJ mutant in the presence of zinc (Fig 6B). Thus, the zntA knockout lost the zinc resistance caused by the rpmJ knockout, whereas the zitB knockout did not affect the zinc resistance. These results suggest that the zntA gene is required for the zinc resistance caused by the rpmJ knockout.  Table). Knockout of several ribosomal proteins leads to a zinc resistance phenotype E. coli has 7 nonessential ribosomal proteins other than RpmJ. We examined whether knockout of these nonessential ribosomal proteins leads to zinc resistance as in the case of the rpmJ knockout. Knockout of rplA, rpmE, rpmI, and rpsT also caused zinc resistance (Fig 7). The results suggest the existence of some conserved zinc resistance mechanisms among the gene knockout mutants of ribosomal proteins.

Discussion
The present findings revealed that knocking out ribosomal protein RpmJ confers zinc resistance to E. coli. The rpmJ mutant had a low concentration of intracellular zinc, which is probably caused by zinc efflux through zntA. RNA sequence analysis revealed that the rpmJ mutant decreased expression of iron-sulfur cluster synthesis genes. Furthermore, knocking out other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, led to zinc resistance in E. coli. This study is the first to reveal that ribosomal protein deficiency causes E. coli resistance to zinc. By constructing gene knockout mutants of zinc efflux pumps, we revealed that the zntA gene is required for the zinc resistance caused by the rpmJ knockout. However, RNA sequence analysis did not reveal differential expression of zntA in the rpmJ mutant. Because the RNA sequence analysis used RNA samples prepared under a no-zinc condition, it is possible that zntA was differentially expressed in the rpmJ mutant under excess zinc conditions. Another possibility is that ZntA protein expression or the activity is changed in the rpmJ mutant. Thus, we assume that the rpmJ knockout leads to zinc resistance by upregulating a zntA-dependent efflux of zinc in some unidentified mechanism. In addition, RNA sequence analysis identified that the rpmJ mutant had decreased expression of genes involved in the synthesis of iron-sulfur clusters. The downregulated expression of iron-sulfur cluster synthesis genes might be involved in the zinc resistance of the rpmJ mutant.
The rpmJ mutant was sensitive to protein synthesis inhibitors, and exhibited altered translation fidelity and increased expression of ribosomal subunit genes. RNA sequence analysis also revealed altered expression of many genes other than ribosome-related genes in the rpmJ mutant, including respiratory genes, metabolic genes for amino acids and DNA, and stress response genes. These findings suggest that structural abnormalities or functional alterations of ribosomes in the rpmJ mutant are sensed by some transcriptional regulators, leading to differential transcription of various genes. Ribosomal proteins are able to repress their own gene translation [20], but the effects on other genes are not known. The stringent response is a wellknown phenomenon that regulates the transcription of many genes when amino acids are limited and translation is inhibited [21]. In the stringent response, tRNA without an amino acid enters into the ribosome A-site and activates RelA protein, a synthase of ppGpp. ppGpp produced by RelA activates the transcription of various genes [22,23]. The altered structure or dysfunction of ribosomes in the rpmJ mutant may result in activation of RelA to induce the expression of various genes. The molecular trigger that induces gene expression changes and interrelationships between the altered gene expressions should be investigated in future studies.
Previous studies demonstrated that 8 ribosomal proteins interact with zinc [24,25]. Among the 5 ribosomal proteins whose knockout leads to zinc resistance, RpmJ and RpmE interact with zinc [26]. Under zinc-limited conditions, RpmJ and RpmE are released from ribosomes and supply zinc by self-degradation, and subsequently YkgO and YkgM, non-zinc binding paralogs of RpmJ and RpmE, form complex with ribosome [27][28][29][30][31]. In contrast, RplA, RpmI, and RpsT, whose knockout leads to zinc resistance, do not interact with zinc and do not function in zinc homeostasis. Thus, the capacity of the ribosomal protein to interact with zinc is not related to the zinc resistance conferred by the knockout of the ribosomal protein. We speculate that some abnormalities of the ribosomal structure and function are conserved among the ribosomal protein mutants that showed zinc resistance. The present study also demonstrated that knockout of rimP, involved in 30S ribosome maturation [15], and tufA, involved in ribosomal peptide elongation [16], leads to zinc resistance in E. coli. The rimP-and tufAknockout mutants could have ribosomal abnormalities and may have the same zinc-resistant mechanisms as the ribosomal protein mutants. Further studies are needed to clarify the molecular mechanisms underlying zinc resistance by investigating ribosomal structure and function in the zinc-resistant mutants identified in this study.

Evaluation of bacterial resistance to antimicrobial substances
To measure bacterial resistance to zinc and antibiotics, autoclaved LB agar medium was mixed with ZnSO 4 �7H 2 O (Nacalai Tesque, Kyoto, Japan) or antibiotics and poured into square plastic dishes (Eiken Chemical, Tokyo, Japan). E. coli overnight cultures were serially diluted 10-fold in 96-well microplates, and 5 μl of the diluted bacterial solution was spotted onto the LB agar plates supplemented with drugs. The plates were incubated at 37˚C for 1 day and colonies were photographed using a digital camera. The MIC values for zinc were determined by spotting bacterial cell suspension (10 5 CFU) onto LB plates supplemented with zinc and incubating the plates overnight at 37˚C.

Genetic manipulation
Gene knockout mutants were constructed by phage transduction using phage P1 vir from the gene knockout mutants in the Keio collection as donor strains to the BW25113 strain as the recipient strain (Table 2). Double knockout mutants were also constructed by phage transduction using phage P1 vir from the gene knockout mutants in the Keio collection as donor strains to the rpmJ mutant, whose Kan r marker was deleted, as the recipient strain. To construct a plasmid carrying the rpmJ gene, a DNA fragment encoding the rpmJ gene was amplified by polymerase chain reaction (PCR) using primer pairs (rpmJ_F_XbaI_2nd and rpmJ_R_Hin-dIII_2nd; Table 3) from genomic DNA of the BW25113 strain as a template. The amplified DNA fragment was cloned into XbaI and HindIII sites of pMW118, resulting in pMW118-rpmJ. Amino acid substitution mutations were introduced into pMW118-rpmJ by PCR using primer pairs (rpmj_C27S_F and rpmj_C27S_R or rpmj_H33S_F and rpmj_H33S_R; Table 3) and pMW118-rpmJ as a template. Mutations were confirmed by DNA sequencing.

Dual-luciferase assay
The wild-type E. coli strain and rpmJ knockout mutant were transformed with plasmids [pQE-Luc(UGA), pQE-Luc(UAG), pQE-Luc(+1), pQE-Luc(-1)] [18] (Table 2). Each transformant was aerobically cultured in LB liquid medium containing 100 μg/ml ampicillin at 37˚C overnight. The overnight culture was inoculated into a 100-fold amount of fresh LB medium. For cells in the no-zinc condition, cells were cultured until OD 600 = 0.5 and then collected. For cells in the zinc condition, cells were cultured until OD 600 = 0.25-0.35 in the no-zinc condition, supplemented with 0.8 mM Zn(II), and then further cultured for 1 h before collecting. The cell pellets were suspended in 200 μl buffer (50 mM HEPES-KOH [pH7.6], 100 mM KCl, 10 mM MgCl 2 , 7 mM β-mercaptoethanol, 400 μg/ml lysozyme). The cell sample was then subjected to freezing and thawing using liquid nitrogen and centrifuged at 15,000 rpm for 15 min at 4˚C. The centrifuge supernatant was mixed with an equal volume of Firefly luciferase

Measurement of intracellular zinc concentration
Zinc concentrations were measured according to a previously reported method [19]. Briefly, 100 μl of E. coli overnight cultures were spread on agar plates supplemented with no zinc, 0.6 mM Zn(II), or 1.2 mM Zn(II), and cultured overnight at 37˚C. The cells were suspended in phosphate buffered saline and the OD 600 value was adjusted to 0.5. The sample was centrifuged, the bacterial pellet was washed 5 times with cold phosphate buffered saline, and 100 μl of 50% HNO 3 was added. The sample was heated at 65˚C overnight. The HNO 3 concentration was adjusted to 5% and the zinc concentration was determined by ICP-MS (Agilent7500cx, Agilent Technologies). The concentrations of other metal elements were measured as well (S1 Fig).

RNA-sequence analysis
Total RNA of E. coli was extracted according to a previously described method [32] with minor modifications. E. coli overnight culture (50 μl) was inoculated into 5 ml LB medium and aerobically cultured at 37˚C. When the OD 600 of the culture reached 0.7, 1.8 ml of culture was vortex-mixed with 200 μl of 5% phenol in ethanol, chilled in ice water for 5 min, and centrifuged at 21,500×g for 2 min. The bacterial precipitate was frozen in liquid nitrogen and stored at −80˚C for 2 h. The precipitate was dissolved in 200 μl lysis buffer (TE buffer, 1% lysozyme, 1% sodium dodecyl sulfate) and incubated at 65˚C for 2 min. The sample was subjected to RNA extraction using an RNeasy minikit (Qiagen) according to the manufacturer's protocol. rRNA was removed from the total RNA using a NEBNext rRNA depletion kit (NEB), and RNA was converted to a DNA library using a TruSeq stranded total RNA kit (Illumina). RNA sequencing was performed using a NovaSeq 6000 system (Illumina), and at least 4 billion base sequences of 100-base paired-end reads were generated per sample. The data were analyzed using CLC Genomics Workbench software (version 11.0). The reads were mapped to a reference genome of the E. coli W3110 strain (NCBI reference sequence NC_007779.1), and the reads per kilobase of transcript per million mapped reads (RPKM) were compared between the wild-type strain and the rpmJ mutant. The experiment was independently performed twice to identify the genes for which the mean values differed by >2-fold between BW25113 and ΔrpmJ and the false discovery rate p value was <0.001. GO analysis was performed using software developed by the European Molecular Biology Laboratory (https://www.ebi.ac.uk/ QuickGO).

Statistical analysis
Differences in dual luciferase assay were evaluated by Student's t test in Excel. Differences in the intracellular zinc concentration by ICP-MS were evaluated by Dunnett's test in GraphPad PRISM software.
Supporting information S1